Our long-term objective is to develop and apply novel methods that will significantly advance our understanding of carbohydrate and lipid metabolism in physiological and pathological states, in particular diabetes. These methods have in common the safe 2H- enrichment of body water with 2H20. We plan to achieve the following seven interrelated specific aims. 1. To quantitate glycogen hydrolysis, cycling, and turnover as a function of liver glycogen content. 2. To measure the rate of de novo glycerol formation via labeling from 2H20, and to define: (i)the role of liver and kidney in glycerol production, fatty acid reesterification, and lipolysis quantitation., and (ii) the source of carbon of glycerol-3-P used by adipose tissue to reesterify fatty acids. 3. To determine the pathway(s) by which 2H20 labels H on C1 of glucose, and to define how this labeling affects calculations of gluconeogenesis and glycogenolysis. 4. To develop a method for quantitating glyconeogenesis, based on 2H-enrichment of body water. 5. To evaluate to what extent the transaldolase reactions affect quantitations of gluconeogenesis and glycogenolysis 6. To apply these techniques to measurements of gluconeogenesis (i) in subjects who are at high risk for NIDDM. (ii) in NIDDM patients, and (iii) in the NIDDM patients then treated with metformin. 7. To apply these techniques to measurements of gluconeogenesis in obese patients. The subjects will ingest 2H2O (to measure the contribution of gluconeogenesis to glucose production from the C5/C2 2H-labeling ratio in glucose), and will be infused with (6,6-2H2) glucose (to measure glucose turnover). The 2H-enrichment at the glucose carbons will be amplified six-fold by incorporating them into hexamethylenetetramine for assay. The contribution of gluconeogenesis will be related to hepatic glycogen content measured by 13C-NMR spectroscopy. Our studies will define the role of gluconeogenesis (i) in diabetic hyperglycemia, (ii) as a possible etiologic factor in the onset of NIDDM, and (iii) in the mechanism of metformin's action. They will help establish whether the propensity of upper body obese subjects to insulin resistance, NlDDM, and cardiovascular disease is related to increased gluconeogenesis. Also, the direct conversion of glycogen to glucose may prove to be an important regulator of glycogen content. Our studies will shed new light on how glucose and lipid metabolism in adipose tissue adapts to fasting.